The long term objectives of this proposal are to establish the metabolic pathways of glycosphingolipids and determine their function in developing brain. Two aspects will be investigated in the present proposal: axonal transport of gangliosides; and the role of psychosine in pathogenesis of Krabbe disease and in its animal counterpart, the twitcher mouse. Gangliosides are concentrated in plasma membrane of nerve cells, in particular at synaptic endings, and assumed to play important roles in nerve cell development, maintenance, repair, and function. They are transported from their site of synthesis in neuronal cell soma to the synaptic nerve ending by axonal transport. Since axonal transport reflects neuronal function, in Specific Aim 1, transport of gangliosides will be studied in a variety of conditions in the optic system of goldfish and of neurological mutant mice. A simple, fast, sensitive HPLC method for quantitating individual gangliosides as their 2,4-dinitrophenylhydrazides has been developed in this laboratory, and will be used to characterize ganglioside transport. For example, are individual gangliosides transported at different rates and are these rates affected by perturbation of the nervous system, such as by anesthetics or neurotoxins, or in neurological mutant mice, or during axonal regeneration? Are gangliosides synthesized and/or modified in the nerve endings from precursors such as CMP-sialic acid? Such studies should aid understanding of the role of gangliosides in the nervous system, particularly in regeneration and repair. Specific Aim 2 concerns the toxicity of psychosine, which accumulates in some tissues of patients with Krabbe disease and the twitcher mutant mouse, both of which show deficient cerebroside beta- galactosidase. With our new, highly sensitive method, cellular and subcellular localization of the accumulated psychosine will be determined in the brain of the twitcher mouse in attempts to correlate this with pathogenecity. An alternative possibility is that psychosine causes pathogenesis by evoking an immune reaction, possibly via new epitopes resulting from modification of the cerebroside (by removal of fatty acids), or by conjugation of the primary amino group of psychosine with a native protein, which then becomes immunogenic. Understanding the mechanism of cytotoxicity of psychosine may provide information for better management and eventually therapy for patients with Krabbe disease.